A Functional Kinase Short Interfering Ribonucleic Acid Screen Using Protease-Activated Receptor 2-Dependent Opening of Transient Receptor Potential Vanilloid-4

摘要

Protease-activated receptor 2 (PAR_(2)) is a proinflammatory G-protein coupled receptor (GPCR) that is activated by inflammatory proteases, and its activation initiates signaling pathways that modulate the nonselective cation channel transient receptor potential vanilloid-4 (TRPV4). PAR_(2)-dependent opening of TRPV4 has been attributed to kinase activation, but the identity of the responsible enzymes is unknown. Deciphering the signaling pathways involved in the PAR_(2)-dependent opening of TRPV4 may yield new targets for pain treatment. This study has identified specific kinases that are involved in opening TRPV4, using a selective screen of short interfering ribonucleic acid (siRNA) SMARTpools, which individually targeted all human kinases, in human embryonic kidney 293 (HEK293) cells that stably express inducible TRPV4. This screen is unique because it uses a real-time assay measuring intracellular calcium with Fura-2AM dye. From the primary screen, subsequent confirmation screen, and on-target messenger ribonucleic acid expression analysis, we identified two kinases as crucial to the PAR_(2)-dependent opening of TRPV4 in HEK293 cells, mitogen-activated protein kinase 13 and with no lysine kinase 4. In conclusion, this study describes a powerful new application of siRNA knockdown to identity signaling molecules that are responsible for the PAR_(2)-dependent opening of TRPV4, which will help elucidate this signaling process .